A new technique has been developed for assay of clotting factors, using solid-phase fibrinogen and labelled fibrinogen together as a substrate for assay of thrombin generated from the clotting cascade. When thrombin cleaves both forms of fibrinogen to fibrin, the labelled fibrin in solution associates with the solid phase. This technique, which we have named "enzyme-linked coagulation assay" or ELCA, is more sensitive than existing methodologies and can conveniently be modified to measure any clotting factor of interest. We have developed this technique using microtiter plates coated with fibrinogen as the solid phase, and peroxidase-fibrinogen in solution as the labelled fibrinogen. Because pipetting equipment and plate readers designed for ELISA assays in microtiter plates can be used in this assay, special equipment does not have to be designed for performing this assay. The objective of this project is to develop kits containing appropriate mixtures of reagents for assay of antithrombin III, factors II, X, VIII and IX, and protein C. The assay will be designed using a single format for dilution of plasmas and addition of reagents, and software will be developed for analysis of data transferred directly to a microcomputer from the plate reader. This technique, when developed in kit form for the assay of specific factors, should improve the diagnosis of a number of diseases for which clotting factor assay is an important component in disease management.